INTIMP-03661; No of Pages 8 International Immunopharmacology xxx (2015) xxx–xxx

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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp

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Article history: Received 21 January 2015 Received in revised form 7 April 2015 Accepted 13 April 2015 Available online xxxx

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Keywords: Andrographolide sodium bisulfite Indomethacin Gastric ulcer Antioxidant Apoptosis Rats

School of Chinese Medicines, Guangzhou University of Chinese Medicine, Guangzhou 510006, PR China The First Affiliated Hospital of Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510405, PR China Dongguan Mathematical Engineering Academy of Chinese Medicine, Guangzhou University of Chinese Medicine, Dongguan 523808, PR China d The Second Affiliated Hospital of Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510120, PR China

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Andrographolide sodium bisulfite (ASB), a water-soluble sulfonate of andrographolide has been shown to possess anti-inflammatory, antipyretic and analgesic activities. However, there is no report on the gastroprotective effect of ASB against indomethacin-induced gastric ulcer. Here we investigated the possible anti-ulcerogenic potential of ASB and the underlying mechanism against indomethacin-induced gastric ulcer in rats. The ulcer area, histopathological assessment, contents of gastric mucosal glutathione (GSH), catalase (CAT), superoxide dismutase (SOD), malonaldehyde (MDA) and prostaglandin E2 (PGE2) were examined. In addition, cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2) mRNA expression and immunohistochemical evaluation of HSP70, Bcl-2 and Bax proteins were also investigated. Results indicated that ASB pre-treatment significantly reduced the ulcer areas induced by indomethacin compared with the vehicle group. The gastric levels of GSH, CAT and SOD were markedly increased by ASB while the level of MDA was decreased. In addition, ASB pretreatment significantly promoted the gastric PGE2 levels and up-regulated the COX-1 and COX-2 mRNA expression in comparison with the vehicle group. Immunohistochemistry analysis showed obvious up-regulation of HSP70 and Bcl-2 protein expression while suppression of Bax protein in the gastric tissue of ASB-pretreated group. Taken together, these findings indicated that the gastroprotective effect of ASB might be associated with the improvement of antioxidative status, activation of COX-mediated PGE2 synthesis, down-regulation of Bax proteins and upregulation of Bcl-2 and HSP70 proteins. ASB might have the potential for further development as a promising alternative for antiulcer treatment. © 2015 Published by Elsevier B.V.

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Yu-Hong Liu a,1, Zhen-Biao Zhang a,1, Yi-Feng Zheng a, Hai-Ming Chen a, Xiu-Ting Yu b, Xiao-Ying Chen a, Xie Zhang a, Jian-Hui Xie d, Zu-Qing Su a, Xue-Xuan Feng a, Hui-Fang Zeng b,⁎, Zi-Ren Su a,c,⁎⁎

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Gastroprotective effect of andrographolide sodium bisulfite against indomethacin-induced gastric ulceration in rats

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1. Introduction

Gastric ulcer is one of the common disorders of human gastrointestinal system that affects 10% of the world population [1]. The etiological factors of this disorder include: stress, smoking, nutritional deficiencies, Helicobacter pylori, and frequent and indiscriminate use of nonsteroidal anti-inflammatory drugs (NSAIDs) [2,3]. NSAIDs continue to be in widespread use due to their crucial role as effective antipyretic and analgesic in a wide spectrum of conditions and diseases ranging from common cold to rheumatoid arthritis [4]. However, it is known that the use of NSAIDs is frequently associated with occurrence of gastric mucosal ulcers [5]. Nowadays, there are many different first-line drugs for gastric

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⁎ Corresponding author. Tel.: +86 20 3935 8517; fax: +86 20 3935 8390. ⁎⁎ Correspondence to: Z.-R. Su, School of Chinese Medicines, Guangzhou University of Chinese Medicine, Guangzhou 510006, PR China. Tel.: +86 20 3935 8517; fax: +86 20 3935 8390. E-mail addresses: [email protected] (H.-F. Zeng), [email protected] (Z.-R. Su). 1 These authors contributed equally to this work.

ulcer prevention and treatment, however, most of these drugs have various side effects, and fail to prevent the recurrence of ulcer [6]. The ever-increasing problem of gastric ulcer necessitated the screening and identification of gastroprotective agents from medicinal plants, which might be less toxic and cost-effective in nature. Andrographis paniculata (Burm. f.) Nees (Chuanxinlian or Kalmegh) is widely used as a remedy for laxative, antipyretic, anti-inflammatory, expectorant, digestive and stomachic [7]. The major bioactive compounds include diterpenoids, flavonoids and polyphenols [8,9]. Andrographolide (C20H30O5), the major diterpenoid of A. paniculata, has gastroprotective effect against ulcerogenesis of rats and can inhibit the gastric cancer BGC-823 cell proliferation [10,11]. However, due to poor water solubility, it is not suitable to treat infectious diseases in which etiopathogenesis and symptoms need to be eliminated imperatively. Andrographolide sodium bisulfite (ASB, C20H29O7SNa, chemical structure shown in Fig. 1), a water-soluble sulfonate of andrographolide, is synthesized by an additive reaction with sodium bisulfite. ASB was the main ingredient of Lianbizhi injection, which has been used clinically to treat infectious and parasitic disorders in mainland China [12]. Thus far,

http://dx.doi.org/10.1016/j.intimp.2015.04.025 1567-5769/© 2015 Published by Elsevier B.V.

Please cite this article as: Y.-H. Liu, et al., Gastroprotective effect of andrographolide sodium bisulfite against indomethacin-induced gastric ulceration in rats, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.04.025

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anti-inflammatory and analgesic effects in mice. Safety evaluation indicated that 250 mg/kg of ASB was safe for rats. As for rats, administration of 10, 20, 40 and 80 mg/kg of ASB for 7 days was found to display obvious gastroprotective effect against indomethacin-induced gastric ulcer in our pilot trial. At the same time, considering the dosage of Andrographics paniculata used in Chinese medicine, 40 mg/kg of ASB was selected as the highest test dose.

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2.4. Indomethacin-induced gastric mucosal damage

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All rats were fasted for 24 h prior to the last day of treatment. One hour after the last drug administration, rats were fed orally with indomethacin (50 mg/kg body weight) to induce acute ulcer, while the intact group received water only. Four hours after indomethacin administration, all rats were euthanized and the stomach was removed and opened along the greater curvature, and rinsed with ice-cold saline to remove the gastric contents and blood clots. The stomach was stretched on a piece of glass pane with the mucosal surface facing up and pressed with another glass pane. The stomach was photographed with a digital camera, and the ulcer area (mm 2 ) was measured using an image analyzer software, Image J, and the inhibition percentage was calculated by the following formula as reported [16] with slight modifications: The inhibition percentage was calculated: [(UAcontrol − UAtreated) / UAcontrol] × 100%. Thereafter, 0.5 cm × 0.5 cm of gastric tissue was taken from each stomach and immersed in 4% neutral formalin for histological evaluation, while 1.5 cm × 1.5 cm × 0.2 cm of gastric tissue was taken from each stomach and immersed in the 4% neutral formalin for immunohistochemical evaluation. The rest of the gastric tissue was stored at −80 °C for biochemical determinations and real-time reverse transcription polymerase chain reaction (RT-qPCR).

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2. Materials and methods

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2.1. Animals

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Sprague Dawley (SD) rats (180–220 g) were obtained from the Laboratory Animal Services Centre of Guangzhou University of Chinese Medicine. The animals were maintained on a 12-h light/12-h dark cycle under room temperature (22 ± 2 °C) and humidity (50 ± 10%) and fed with standard forage and clean water ad libitum. All studies were conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.

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2.2. Drugs and chemicals

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Andrographolide sodium bisulfite (ASB) was purchased from Wuxi Zhongkun Biochemical Technology Co., Ltd., (Wuxi, China). As the positive control, lansoprazole (LSZ) tablets were supplied by Jiangsu Kanion Pharmaceutical Co., Ltd., (Jiangsu, China). Indomethacin was obtained from Sigma-Aldrich. Ultrapure water was purified by using a Milli-Q gradient water purification system (Millipore, Bedford, MA, USA). All the other chemicals and reagents were of commercially available analytical grade.

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The rats were allowed for 7 days in the pre-experimental period to adapt to the laboratory conditions. Rats were randomly divided into six experimental groups, each containing six animals. The intact and vehicle groups received (i.g.) 0.5% sodium carboxymethylcellulose throughout the course of the experiments. The prevention groups received (i.g.) different doses of ASB (10, 20 and 40 mg/kg, dissolved in 0.5% sodium carboxymethylcellulose) and lansoprazole (30 mg/kg, reference drug, dissolved in 0.5% sodium carboxymethylcellulose) respectively for a period of 7 days. All drugs were administered once daily. In our preliminary dose responsive experiment, oral pretreatment with 1, 3 and 5 mg/kg of ASB to rats for 7 consecutive days, an ordinary administration span three weeks shorter than the administration period reported by Saranva et al. [10], failed to show significant reduction in indomethacin-induced gastric ulcer. According to some previous studies [13–15], 40, 80 and 160 mg/kg of ASB were adopted for its possible

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there is no report on the anti-ulcerogenic activity of ASB. Hence, this study was undertaken to evaluate the possible gastroprotective effect of ASB against indomethacin-induced gastric ulcer in rats and the potential underlying mechanism.

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Fig.1. The structure of andrographolide sodium bisulfate.

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2.5. Histopathological assessment

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For histological assessment, the gastric tissue dehydrated in ascending grades of alcohol and then embedded in paraffin. Sections of 5 μm thick were taken, which were placed onto glass slides and deparaffinized, stained with hematoxylin–eosin (H&E). All tissue sections were examined under a light microscope. The specimens were assessed according to the criteria as previously reported [17].

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2.6. Measurement of catalase (CAT), total glutathione (GSH), superoxide 148 dismutase (SOD) and malonaldehyde (MDA) level 149 Stomach tissues stored at −80 °C were weighed, minced by forceps, and homogenized in 4 mL Tris-buffer (20 mM, pH = 7.5) on ice using Ultra Turrax Homogenizer (IKA, Germany). Then the resulting homogenates were centrifuged at 12,000 g at 4 °C for 10 min to obtain the supernatants. The supernatants were used to determine the levels of CAT, GSH, MDA and SOD [15,18,19].

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2.6.1. CAT assay CAT activity was determined according to the ammonium molybdate spectrophotometric method, based on the fact that ammonium molybdate can rapidly terminate the H2O2 degradation reaction catalyzed by CAT and react with the H2O2 to generate a yellow complex that is monitored by the absorbance at 405 nm. The experimentation was launched according to the manufacturer's instructions (Jiancheng Company, Nanjing, China).

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2.6.2. GSH assay GSH activity was measured according to the method which is based on the principle that dithio-bis-nitrobenzoic acid (DTNB) can react with sulfhydryl compound to generate a yellow complex. The reaction mixture was determined colorimetrically at 420 nm by using a Multiskan

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Please cite this article as: Y.-H. Liu, et al., Gastroprotective effect of andrographolide sodium bisulfite against indomethacin-induced gastric ulceration in rats, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.04.025

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2.6.3. MDA assay MDA activity was evaluated by the method of thiobarbituric acid reacting substance (TBARS). The whole experiment process was completed according to the manufacturer's instructions (Jiancheng Company, Nanjing, China). Briefly, MDA reacted with TBARS at 100 °C and formed a red complex which could be measured by the absorbance at 532 nm.

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2.7. Determination of prostaglandin E2 (PGE2) levels

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Stomach tissues stored at −80 °C were weighed, minced by forceps, and homogenized in 1 mL Tris-buffer (20 mM, pH = 7.5) on ice using Ultra Turrax Homogenizer (IKA, Germany). Then the resulting homogenates were centrifuged at 12,000 g at 4 °C for 10 min to obtain the supernatants. The supernatants were used for determination of PGE2 by using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions (R&D Systems, Abingdon, UK). Curtly, diluted standards or samples were added to 96-well plates precoated with affinity purified polyclonal antibody specific for rat PGE2. The wells were added with enzyme-linked polyclonal antibodies and incubated at 37 °C for 60 min, followed by final washes for 5 times. The optical densities were measured at 450 nm and results were expressed as pg/mg tissue.

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2.8. Determination of COX-1 and COX-2 mRNA by RT-qPCR

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Samples of the gastric tissue were obtained to quantify cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) mRNA using RT-qPCR [20,21]. Total RNA was extracted by using RNAiso Plus

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2.6.4 . SOD assay SOD activity was measured according to the xanthine oxidase method, on the basis of its ability to inhibit the oxidation of hydroxylamine by the xanthine–xanthine oxidase system. It could be accomplished according to the manufacturer's instructions (Jiancheng Company, Nanjing, China). Curtly, xanthine and xanthine oxidase (XOD) generate superoxide radicals that reacted with hydroxylamine to form an amaranth nitrite which could be determined by the absorbance at 550 nm.

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Reagent (Takara, Japan) and according to the protocol provided by the manufacturer. Then total RNA (2 μg) was reverse-transcribed into cDNA using BestarTM qPCR RT Kit with gDNA eraser (DBI Bioscience, Germany) following the manufacturer' s instructions. The PCR was performed in a system of Agilent Stratagene Mx3000P fluorescence quantitative (Agilent Stratagene, USA) using DBI Bestar® SybrGreen qPCRmasterMix (DBI Bioscience, Germany) as recommended by the manufacturer. The fluorescent quantity PCR conditions were as follows: pre-denaturation at 94 °C for 2 min, then 40 cycles of 94 °C for 20 s, 58 °C for 20 s and 72 °C for 20 s. Melting curve program was added afterwards. Primers were as follows: COX-1, forward, 5′-CCCACCTTCCGT AG AACAGG-3′, and reverse, 5′-GAGCAACCCAAACACCTCCT-3′; COX-2, forward, 5′-CATTGACCAGAGCAGAGAGAT-3′, and reverse, 5′-CTTTCT CTCCTGTAAGT TCTT-3′; GAPDH, forward, 5′-CCTCGTCTCATAGACAA GATGGT-3′, and reverse, 5′-GGGTAGAGTCATACTGGA ACATG-3′. In PCR data, the fold change in cDNA relative to the GAPDH endogenous control was calculated by 2-ΔΔCt method [22]. The whole experiment was carried out according to the MIQE guidelines [23].

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Fig.2. Effect of ASB on the macroscopic appearance of the gastric mucosa in the indomethacin-induced gastric ulcer in rats. (A) Intact group; (B) vehicle group: banded hemorrhagic necrosis of gastric mucosa caused by indomethacin. (C) Lansoprazole group: lansoprazole (30 mg/kg) + indomethacin; (D) ASB (10 mg/kg) + indomethacin; (E) ASB (20 mg/kg) + indomethacin; (F) ASB (40 mg/kg) + indomethacin; (D–F): different degrees of petechial hemorrhagic necrosis could be found.

Fig.3. Effect of ASB on the gastric ulcer area (mm2) of rats' stomachs (n = 6) in indomethacin-induced gastric ulcer. Rats were administrated intragastrically with lansoprazole (30 mg/kg) or ASB (10, 20 and 40 mg/kg) prior to intragastrical administration of indomethacin (50 mg/kg). The results were expressed as mean ± SEM and analyzed by ANOVA followed by Dunnett's test. **P b 0.01, *P b 0.05 versus the vehicle group. Student's t-test was performed for comparing intact and vehicle groups, ##P b 0.01. (LSZ: lansoprazole).

Please cite this article as: Y.-H. Liu, et al., Gastroprotective effect of andrographolide sodium bisulfite against indomethacin-induced gastric ulceration in rats, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.04.025

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Fig.4. Effect of ASB on histological assessment in indomethacin-induced gastric ulcer of rats (HE staining, magnification 100×). (A) Intact group: Rats pretreated with vehicle. (B) Vehicle group: * indicates lesion on the surface epithelium with disrupted glandular structure, and arrow indicates extensive edema of submucosal layer. (C) Lansoprazole group: lansoprazole (30 mg/kg) + indomethacin; (D) ASB (10 mg/kg) + indomethacin; (E) ASB (20 mg/kg) + indomethacin; (F) ASB (40 mg/kg) + indomethacin.

using the software called Image-Pro Plus (IPP, version 6.0, Media 250 Cybernetics, Inc.). IOD reflects the total expression of proteins in 251 selected area. 252

2.9. Immunohistochemical evaluation

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Gastric tissue sections (prepared as mentioned in Section 2.4.) were washed with water and dehydrated with graded alcohol. Then xylene and alcohol were used to hyalinize the tissues. After embedding in paraffin and cutting into slices, we could get the paraffin sections of 5 μm thickness. The sections were heated at 60 °C for 1 h in an oven, then deparaffinized in xylene and rehydrated using graded alcohol. 3% H2O2 solution was added to the stomach tissue biopsies in order to block and inactivate endogenous peroxidase, and sections were incubated at 37 °C for 10 min. After that, the sections were washed by Tris-buffered saline (TBS, pH = 7.6) three times and blocked with 0.01% D-biotin for pretreatment with the heat-induced epitope retrieval method. The sections were further blocked with 5% bovine serum albumin for 0.5 h to minimize nonspecific antibody blinding and incubated with appropriate dilution of primary antibodies for 1 h at room temperature or overnight at 4 °C. Immunohistochemical staining of HSP70, Bax and Bcl-2 proteins, was performed according to the manufacturer's protocol (Santa Cruz Biotechnology, Inc. American) [24,25]. Monoclonal anti-rat HSP70 antibody, anti-rat Bax antibody and anti-rat Bcl-2 antibody were used at a dilution of 1:500, 1:500 and 1:500 respectively. In staining of HSP70 proteins, tissue sections were pretreated by boiling in Tris-buffer containing EDTA (pH = 9) to retrieve the antigen and incubated with the primary antibody for 1 h at room temperature. In staining of Bax or Bcl-2 proteins, tissue sections were pretreated by boiling in Tris-buffer containing EDTA (pH = 6) for 0.5 h to retrieve the antigen and incubated with the primary antibody for 1.5 h at room temperature. The sections were photographed with digital camera, and the integrated optical density (IOD) [26–28] was analyzed

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Table 1 Effect of ASB on CAT, GSH, MDA and SOD activities in the gastric tissue of indomethacin-treated rats (n = 6).

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The results are expressed as mean ± SEM with a value of P b 0.05 considered statistically significant. Statistical evaluation was performed by using a one-way analysis of variance (ANOVA) followed by Dunnett's test for multiple comparisons. Student's t-test was used in the case of comparison between two groups. All analyses were made with Statistical Product and Service Solutions (SPSS) software (version 20.0).

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Representative gross appearance of gastric mucosa for each group is shown in Fig. 2 and the ulcer areas are depicted in Fig. 3. Nearly no macroscopic lesions were observed in the intact group (1.12 ± 0.1 mm2, Fig. 2A). Administration of indomethacin (Fig. 2B) caused a significant increase in the ulcer area (22.89 ± 1.85 mm2) when compared with the intact group. For the lansoprazole-treated group (Fig. 2C), the ulcer area was significantly decreased (P b 0.01) with an average of 5.80 ± 1.14 mm2 (74.67% inhibition). Pretreatment with ASB at different dosages (10, 20 and 40 mg/kg) led to decrease of ulcer area in a dose-dependent manner, with the minimum ulcer area (6.91 ± 0.82 mm2) and the highest inhibition (69.83%) observed at the dose of 40 mg/kg (Fig. 2F). The ulcer area of ASB at dose of 20 (Fig. 2E) and

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Intact Vehicle Lansoprazole ASB ASB ASB

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SOD (units/mg protein)

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19.13 ± 0.42 10.34 ± 0.98## 18.80 ± 0.97** 14.03 ± 0.40 16.36 ± 0.69** 18.23 ± 1.26**

41.61 ± 2.81 19.77 ± 0.45## 33.83 ± 1.49** 23.56 ± 0.76* 25.46 ± 0.63** 33.58 ± 1.75**

7.53 ± 0.57 18.78 ± 0.51## 11.16 ± 0.47** 13.09 ± 1.88* 11.62 ± 2.01** 11.11 ± 0.23**

40.94 ± 1.17 29.59 ± 1.82## 42.45 ± 2.40** 37.29 ± 1.09* 38.56 ± 1.22** 41.12 ± 1.81**

The results were expressed as mean ± SEM and analyzed by ANOVA followed by Dunnett's test. **P b 0.01, *P b 0.05 versus the vehicle group. Student's t-test was performed for comparing intact and vehicle groups, ##P b 0.01.

Please cite this article as: Y.-H. Liu, et al., Gastroprotective effect of andrographolide sodium bisulfite against indomethacin-induced gastric ulceration in rats, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.04.025

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10 mg/kg (Fig. 2D) was 8.49 ± 1.14 and 10.80 ± 1.74 mm2 (62.91% and 52.83% inhibition), respectively.

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Fig. 4 demonstrated the results of histological analyses of the gastric mucosa. The vehicle group (Fig. 4B) showed severe damages to the gastric epithelium and edema of the submucosal layer. Pretreatment with ASB (40 mg/kg, Fig. 4F) and lansoprazole (Fig. 4C) had comparatively better protection of the gastric mucosa as seen by least mucosal damage and mildest submucosal edema when compared to the vehicle group. Rats pretreated with ASB at 20 (Fig. 4E) and 10 mg/kg (Fig. 4D) showed improved alternations, displaying less mucosal injury and milder edema respectively.

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3.3. Effects of ASB on gastric CAT, GSH, MDA, and SOD levels

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As shown in Table 1, CAT, GSH and SOD contents decreased markedly (P b 0.01) in the gastric tissue of the vehicle group respectively while MDA activity increased significantly (P b 0.01) in comparison with the intact group. Pretreatment with ASB (20 and 40 mg/kg) could relieve the oxidant status by significantly enhancing CAT, GSH and SOD activities and reducing MDA level (P b 0.01) in the gastric mucosa when compared with the vehicle group. ASB at 10 mg/kg increased the CAT, GSH and SOD (P b 0.05) contents while it decreased MDA (P b 0.05) content in comparison with the vehicle group, though there was no statistical significance (P N 0.05) on the CAT level.

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3.4. Effect of ASB on gastric PGE2 level

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As shown in Table 2, the PGE2 level was significantly (P b 0.01) lower in the vehicle group than that of the intact group. Pretreatment with ASB markedly (at least P b 0.05) restored the depressed PGE2 level in a dose-dependent manner, which was suppressed due to indomethacin administration. Of which, rats pretreated with ASB (40 mg/kg) showed significant increment in PGE2 content when compared with the vehicle group (P b 0.01), which was even comparable to the reference drug lansoprazole.

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3.5. Expression of COX-1 and COX-2 mRNA

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As shown in Fig. 5, the result of RT-qPCR revealed that administration with indomethacin could lead to down-regulation of transcript COX-1 and COX-2 expressions (1.64-fold and 3.13-fold, respectively) markedly. On the other hand, COX-1 and COX-2 mRNA expressions in rats pretreated with ASB (40 mg/kg) were significantly enhanced (1.39-fold, P b 0.05; 2.43-fold, P b 0.01, respectively) in a dose-related manner when compared to the vehicle group. Administration with lansoprazole also resulted in higher expressions of COX-1 and COX-2 mRNA (1.41-fold, P b 0.05; and 2.48-fold, P b 0.01, respectively).

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3.6. Immunohistochemistry of HSP70, Bax and Bcl-2 proteins

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Results of immunohistochemical staining were shown in Figs. 6 and 7. Administration with indomethacin caused down-regulation of HSP70 (IOD: 0.71 ± 0.06, Fig. 7A) and Bcl-2 (IOD: 1.44 ± 0.23, Fig. 7C) protein expressions in the gastric tissue while the expression of Bax protein (IOD: 16.23 ± 0.81, Fig. 7B) was up-regulated. On the contrary, ASB pre-treatment enhanced the expressions of HSP70 (10 mg/kg: IOD 6.09 ± 0.74; 20 mg/kg: IOD 6.69 ± 0.72; 40 mg/kg: IOD 8.89 ± 0.73; Fig. 7A) and Bcl-2 protein (10 mg/kg: IOD 2.87 ± 0.28; 20 mg/kg: IOD 3.02 ± 0.33; 40 mg/kg: IOD 14.27 ± 0.87; Fig. 7C) and reduced Bax protein expression (10 mg/kg: IOD 4.66 ± 0.57; 20 mg/kg: IOD 2.81 ± 0.50; 40 mg/kg: IOD 1.18 ± 0.34; Fig. 7B).

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Table 2 t2:1 Effect of ASB on the indomethacin-induced changes in PGE2 content in gastric tissue t2:2 homogenate (n = 6). t2:3

Intact Vehicle Lansoprazole ASB ASB ASB

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t2:5

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65.78 ± 1.41 50.81 ± 2.18## 65.21 ± 2.71** 63.61 ± 3.34* 64.85 ± 3.20** 66.15 ± 2.83**

t2:6 t2:7 t2:8 t2:9 t2:10 t2:11

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In this study, we investigated the gastroprotective effects of three doses (10, 20 and 40 mg/kg) of ASB and lansoprazole (30 mg/kg) against indomethacin induced gastric ulceration in rats. Macroscopic analyses showed that indomethacin caused considerable gastric ulceration. Indomethacin-induced ulcers in rats have become a standard model for assessing the protective effects of compounds against NSAID-induced gastric damage, in which augmented oxidative stress (lipid peroxidation, protein oxidation, hydroxyl radical generation) was involved in the pathogenic mechanisms [29,30]. In previous studies [31,32], it was found that NSAID-induced gastric ulcer was caused mainly by inhibiting COX-mediated prostaglandin (PG) synthesis. Meanwhile, apoptosis also played an important role in generating gastric ulcers [33]. Therefore, the antioxidant status, COX-mediated PG synthesis and anti-apoptosis proteins were investigated to unravel the possible mechanism underlying the gastroprotective effects of ASB against indomethacin-induced gastric ulcer in rats. Experimental studies have indicated that reactive oxygen species (ROS) play an important role in NSAID-induced gastric ulcers [34]. NSAIDs such as indomethacin, could initiate lipid peroxidation by oxidizing and cause damage by producing ROS. Organisms have enzymatic and non-enzymatic defenses, including SOD, GSH and CAT, which could reduce or prevent the injury of gastric tissue caused by ROS [35]. GSH, an important intracellular antioxidant, protects gastric mucosa from free radical-induced tissue damage [36]. SOD protects stomach tissue from damaging by converting the highly reactive radical superoxide (O− 2 ) into less reactive peroxide (H2O2), which can be destroyed by CAT to form water and oxygen [37]. In addition, other studies revealed

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PGE2 (ng/g protein)

The results were expressed as mean ± SEM and analyzed by ANOVA followed by t2:12 Dunnett's test. **P b 0.01, *P b 0.05 versus the vehicle group. Student's t-test was performed t2:13 for comparing intact and vehicle groups, ##P b 0.01. t2:14

4. Discussion

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Fig.5. Effect of ASB on mRNA expressions of COX-1 and COX-2 in gastric mucosa of indomethacin-induced rats (n = 6). Rats were administrated intragastrically with lansoprazole (30 mg/kg) or ASB (10, 20 and 40 mg/kg) prior to intragastrical administration of indomethacin (50 mg/kg). The results were reported as mean ± SEM and analyzed by ANOVA followed by Dunnett's test. **P b 0.01, *P b 0.05 versus the vehicle group.

Please cite this article as: Y.-H. Liu, et al., Gastroprotective effect of andrographolide sodium bisulfite against indomethacin-induced gastric ulceration in rats, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.04.025

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Fig.6. Immunohistochemical evaluation of HSP70, Bax and Bcl-2 proteins expressions in the stomachs of rats in indomethacin-induced gastric ulcer (magnification 200×). The antigen sites appeared as brown color (arrow). First row: immunohistochemistry staining of HSP70 proteion; second row: immunohistochemistry staining of Bax protein; third row: immunohistochemistry staining of Bcl-2 protein; intact group: rats only pretreated with vehicle; vehicle group: vehicle + indomethacin; lansoprazole: lansoprazole (30 mg/kg) + indomethacin; ASB 10 mg/kg: ASB + indomethacin; ASB 20 mg/kg: ASB + indomethacin; ASB 40 mg/kg: ASB + indomethacin. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig.7. Integrated optical density (IOD) of HSP70, Bax and Bcl-2 protein expressions in the stomachs of rats in indomethacin-induced gastric ulcer (n = 6). Rats were administrated intragastrically with lansoprazole (30 mg/kg) or ASB (10, 20 and 40 mg/kg) prior to intragastrical administration of indomethacin (50 mg/kg). A: IOD of HSP70 protein expression. B: IOD of Bax protein expression. C: IOD of Bcl-2 protein expression. The results were reported as mean ± SEM and analyzed by ANOVA followed by Dunnett's test. **P b 0.01, *P b 0.05 versus the vehicle group. (LSZ: lansoprazole).

Please cite this article as: Y.-H. Liu, et al., Gastroprotective effect of andrographolide sodium bisulfite against indomethacin-induced gastric ulceration in rats, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.04.025

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inhibits apoptosis and Bax protein promotes the apoptosis process [51]. When the expression of Bax protein increases, apoptosis might be induced with forming homodimer. In contrast, when the expression of Bcl-2 protein is enhanced, heterodimer would be competitively formed against the Bax protein. Then the Bcl-2–Bax heterodimer can inhibit the apoptosis induced by Bax–Bax homodimer [52]. It was reported previously that the expression of Bcl-2 protein was enhanced in the stomach tissue in repair stage of acute gastric mucosal injury while the expression of Bax decreased [53]. In the present study, as compared with the vehicle group, pretreatment with ASB exhibited a down-regulation of Bax protein expression and an up-regulation of Bcl-2 protein expression in the gastric mucosal tissue by immunohistochemical staining. It is suggested that ASB might effectively inhibit indomethacin-induced gastric ulcer via regulating equilibrium of expression of Bcl-2 and Bax proteins. On the other hand, the expression of heat shock protein (HSP70) also takes an important part in the intracellular mechanism of gastric protection. HSP70 functions by maintaining normal protein structures and eliminating the damaged ones induced by various external and cellular stresses such as heat intolerance, immune response or oxidative stress [54]. It has been reported that HSP70 can enhance the tolerance of gastric mucosa to NSAIDs, and there is a positive correlation between its protection and concentration [25]. The findings obtained showed remarkable up-regulation of HSP70 expression in ASB-pretreated group as compared to the vehicle. It is therefore hypothesized that the induction of HSP70 synthesis might perform an essential role in enhancing the gastroprotective effect of ASB. In summary, the modulation of Bax, Bcl-2 and HSP70 protein expressions observed in this study might indicate that ASB exerted the gastroprotective effect, at least in part, through the regulation of Bax, Bcl-2 and HSP70 proteins. The results of the present study clearly indicated that ASB displayed gastroprotective effect against indomethacin-induced gastric ulcer in rats. The protection of ASB was shown to be dose-dependent according to the reduction of ulcer areas in gastric mucosa and inhibition of edema in submucosal layers. The underlying mechanisms of its gastroprotective effect might involve the improvement of antioxidative status, activation of COX-mediated PGE2 biosynthesis, suppression of Bax protein expression, and promotion of HSP70 and Bcl-2 protein expressions. ASB might have the potential for further development as a promising alternative against NSAID mediated gastric ulceration.

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an increase in MDA content and decrease in antioxidant levels in gastric mucosa of NSAID-treated rats [38,39]. Similarly, in the present study, the contents of SOD, GSH and CAT in rat stomach tissues of the vehicle group were reduced significantly while the content of MDA was increased markedly. In contrast, ASB pre-treated groups showed increments in SOD, GSH and CAT levels, and decrease in MDA content. In view of our results, it might be concluded that the ability of ASB in reducing the lipid peroxidation (MDA) and restoring the levels of nonenzymatic (GSH) and enzymatic (SOD and CAT) might contribute to its gastroprotective effect by inhibiting oxidative gastric damage. Prostaglandin (PG) is a key molecule that activates the ulcer-healing mechanism, and it is synthesized in the gastric mucosal cells by COX enzymes. It stimulates the secretion of bicarbonates and mucus, promotes ulcer healing, and inhibits the secretion of gastric acid [40]. Inhibition of PG synthesis by indomethacin is known to cause gastric ulcer and exacerbate preexisting gastric ulceration in rodents and humans [41]. PGs can be divided into PGA, PGE, PGF and PGI, of which PGE2 is the most important PGs in the human gastrointestinal tract with the largest amount [42]. Indomethacin was deemed to cause ulcer mainly through suppression of PGE2 synthesis [43]. Cyclooxygenase (COX), target enzyme of NSAIDs, plays a major role in the synthesis of prostaglandin with arachidonic acid. Its isomers include COX-1 and COX-2. Though COX-1 has 60% homology with COX-2, there is an obvious distinction in respect of function [44]. COX-1, a constitutive enzyme which is expressed in tissues and cells constantly, synthesizes prostaglandin to maintain the physiological processes and function of organism normally. Suppression of COX-1 could lead to the decrease of synthesis of prostaglandin, which might reduce the secretion of mucus, lessen mucosal blood flow, delay the repair of cells and damage the gastric mucosal barrier. In addition, COX-2, an inducible enzyme, appears mainly in the pathological situation which is activated by endotoxin, proinflammatory cytokines and stimulating factors in tissue repair. The expression of COX-2 increases in the case of gastric mucosa damage to exert the protection for mucous membrane [45]. Therefore, COX-2 could alleviate the prostaglandin deficiency while COX-1-induced PG synthesis is decreased [46]. Indomethacin is a nonselective inhibitor of both COX-1 and COX-2 which can cause damage in the stomach with a significant decrease in the gastric mucosal PGE2 content. In the present work, the level of PGE2 was reduced markedly in the vehicle group with decreased expressions of COX-1 and COX-2 when compared to the intact group, which was in line with these previous studies. It was possible that PGs converted into the products of oxidation such as 8-iso-PGF2α in the presence of oxidative damage [47]. Besides, oxidative stress could inhibit the COX activity which led to the decrease of PGE2 levels [48]. On the other hand, administration of ASB markedly boosted the depressed PGE2 level as compared to the vehicle group in a dose-dependent manner, with the high dose group being the most significant. Meanwhile, the expressions of COX-1 and COX-2 were up-regulated in the ASB-pretreated group as compared to the vehicle. These results might be associated with the antioxidant activity of ASB which reduced the oxidative stress and eliminated the inhibitory effect on COX expression. And the stimulative effect on COX level might contribute to maintain the level of gastric PGE2 after indomethacin administration. Based on these results, it is perceived that COX-mediated PGE2 biosynthesis might take an important part in the gastroprotective effect of ASB against ulcerative damage caused by indomethacin. Gastric ulcer is one of the common and multiplex diseases in clinical practice, and its pathogenesis is closely related to apoptosis in the gastric mucosal epithelial cells by all kinds of stimulation [49]. The normal state of gastric mucosa is always in equilibrium between the cell death and cell renewal, and gastric lesion is produced when there is an increase in apoptosis and/or suppression of cell proliferation [50]. Expression and regulation of Bcl-2 family proteins is one of the key factors affecting cell apoptosis. Thereinto, Bcl-2 and Bax proteins are important representatives of Bcl-2 family, and their variations play a major role in determining cell life. It is reported that Bcl-2 protein

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The authors declared that there were no conflicts of interest.

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This work was supported by grants from the National Natural Science Foundation of China (No. 81173534), Science and Technology Planning Project of Guangdong Province (No. 2012A080202002 & 2013A022100001), Guangdong International Cooperation Project (No. 2012B050300002), Science and Technological Program for Dongguan's Higher Education, Science and Research, and Health Care Institutions (No. 2012105102009), Ph.D. Programs Foundation of Ministry of Education of China (No. 20134425110009) and Science and Technology Innovation Project of Guangdong Provincial Department of Education (No. 2013KJCX0045), and Central Finance of China in Support of the Development of Local Colleges and University [Educational finance Grant No. 276(2014)], and Science and technology cooperation projects among Hong Kong, Macao and Taiwan (No.2014DFH30010).

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References

478

467 468 469 470 471 472 473 474 475 476 477

[1] M.L. Schubert, Gastric secretion, Curr. Opin. Gastroenterol. 20 (2004) 519–525. 479 [2] M.S. Khazaei, Hossein, Protective effect of Falcaria vulgaris extract on ethanol 480 Q7 induced gastric ulcer in rat, Iran. J. Pharmacol. Ther. 5 (2006) 43–46. 481

Please cite this article as: Y.-H. Liu, et al., Gastroprotective effect of andrographolide sodium bisulfite against indomethacin-induced gastric ulceration in rats, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.04.025

D

P

R O

O

F

[28] P.Y. Zhuang, J. Shen, X.D. Zhu, J.B. Zhang, Z.Y. Tang, L.X. Qin, et al., Direct transformation of lung microenvironment by interferon-alpha treatment counteracts growth of lung metastasis of hepatocellular carcinoma, PLoS One 8 (2013) e58913. [29] Y. Naito, T. Yoshikawa, Oxidative stress involvement and gene expression in indomethacin-induced gastropathy, Redox Rep. 11 (2006) 243–253. [30] K. Ganguly, P. Kundu, A. Banerjee, R.J. Reiter, S. Swarnakar, Hydrogen peroxidemediated downregulation of matrix metalloprotease-2 in indomethacin-induced acute gastric ulceration is blocked by melatonin and other antioxidants, Free Radic. Biol. Med. 41 (2006) 911–925. [31] B.J. Whittle, Gastrointestinal effects of nonsteroidal anti-inflammatory drugs, Fundam. Clin. Pharmacol. 17 (2003) 301–313. [32] D. Lamarque, Pathogenesis of gastroduodenal lesions induced by non-steroidal antiinflammatory drugs, Gastroenterol. Clin. Biol. 28 (3) (2004) C18–C26. [33] M. Nakashita, H. Suzuki, S. Miura, T. Taki, K. Uehara, T. Mizushima, et al., Attenuation of acetic acid-induced gastric ulcer formation in rats by glucosylceramide synthase inhibitors, Dig. Dis. Sci. 58 (2013) 354–362. [34] H. Suleyman, E. Cadirci, A. Albayrak, B. Polat, Z. Halici, F. Koc, et al., Comparative study on the gastroprotective potential of some antidepressants in indomethacininduced ulcer in rats, Chem. Biol. Interact. 180 (2009) 318–324. [35] B. Polat, Y. Albayrak, B. Suleyman, H. Dursun, F. Odabasoglu, M. Yigiter, et al., Antiulcerative effect of dexmedetomidine on indomethacin-induced gastric ulcer in rats, Pharmacol. Rep. 63 (2011) 518–526. [36] H.M. Sidahmed, N.M. Hashim, J. Amir, M.A. Abdulla, A.H. Hadi, S.I. Abdelwahab, et al., Pyranocycloartobiloxanthone A, a novel gastroprotective compound from Artocarpus obtusus Jarret, against ethanol-induced acute gastric ulcer in vivo, Phytomedicine 20 (2013) 834–843. [37] X. Liu, Z. Chen, N. Mao, Y. Xie, The protective of hydrogen on stress-induced gastric ulceration, Int. Immunopharmacol. 13 (2012) 197–203. [38] J.H. Kim, B.W. Kim, H.J. Kwon, S.W. Nam, Curative effect of selenium against indomethacin-induced gastric ulcers in rats, J. Microbiol. Biotechnol. 21 (2011) 400–404. [39] I.Z.A.K.H. Abdallah, G.H. Heeba, Gastroprotective effect of Cordia myxa L. fruit extract against indomethacin-induced gastric ulceration in rats, Life Sci. J. 8 (2011) 433–445. [40] B. Adhikary, S.K. Yadav, K. Roy, S.K. Bandyopadhyay, S. Chattopadhyay, Black tea and theaflavins assist healing of indomethacin-induced gastric ulceration in mice by antioxidative action, Evid. Based Complement. Alternat. 2011 (2011). [41] F. Halter, A.S. Tarnawski, A. Schmassmann, B.M. Peskar, Cyclooxygenase 2implications on maintenance of gastric mucosal integrity and ulcer healing: controversial issues and perspectives, Gut 49 (2001) 443–453. [42] J.L.L.Y. Ren, J.S. Pan, Advances in basic and clinical study of gastric mucosal protection, World Chin. J. Digestol. 13 (2005) 2521–2529. [43] V. Motilva, C. Alarcon de la Lastra, L. Bruseghini, J. Manuel Herrerias, S. SanchezFidalgo, COX expression and PGE(2) and PGD(2) production in experimental acute and chronic gastric lesions, Int. Immunopharmacol. 5 (2005) 369–379. [44] L.J. Crofford, COX-1 and COX-2 tissue expression: implications and predictions, J. Rheumatol. Suppl. 49 (1997) 15–19. [45] R.N. Dubois, S.B. Abramson, L. Crofford, R.A. Gupta, L.S. Simon, L.B. Van De Putte, et al., Cyclooxygenase in biology and disease, FASEB J. 12 (1998) 1063–1073. [46] L. Laine, K. Takeuchi, A. Tarnawski, Gastric mucosal defense and cytoprotection: bench to bedside, Gastroenterology 135 (2008) 41–60. [47] J.R. Avila, C.A. de la Lastra, M.J. Martin, V. Motilva, I. Luque, D. Delgado, et al., Role of endogenous sulphydryls and neutrophil infiltration in the pathogenesis of gastric mucosal injury induced by piroxicam in rats, Inflamm. Res. 45 (1996) 83–88. [48] V. Bertrand, F. Guessous, A.L. Le Roy, B. Viossat, H. Fessi, A. El Abbouyi, et al., Copperindomethacinate associated with zwitterionic phospholipids prevents enteropathy in rats: effect on inducible NO synthase, Dig. Dis. Sci. 44 (1999) 991–999. [49] L. Spyridon, N. Akira, K. Hiromasa, G. Katsutoshi, M. Takao, O. Yoshiki, et al., The development of the endothelin-1-induced gastric ulcer: time sequence analysis of morphologic changes, Int. J. Exp. Pathol. 75 (1994) 345–355. [50] P. Maity, S. Bindu, V. Choubey, A. Alam, K. Mitra, M. Goyal, et al., Lansoprazole protects and heals gastric mucosa from non-steroidal anti-inflammatory drug (NSAID)induced gastropathy by inhibiting mitochondrial as well as Fas-mediated death pathways with concurrent induction of mucosal cell renewal, J. Biol. Chem. 283 (2008) 14391–14401. [51] I. Szabo, A.S. Tarnawski, Apoptosis in the gastric mucosa: molecular mechanisms, basic and clinical implications, J. Physiol. Pharmacol. 51 (2000) 3–15. [52] X.Z. Wang, S.J. Zhang, Y.X. Chen, Z.X. Chen, Y.H. Huang, L.J. Zhang, Effects of plateletderived growth factor and interleukin-10 on Fas/Fas-ligand and Bcl-2/Bax mRNA expression in rat hepatic stellate cells in vitro, World J. Gastroenterol. 10 (2004) 2706–2710. [53] P.C. Konturek, T. Brzozowski, S.J. Konturek, R. Pajdo, J.E. Konturek, S. Kwiecien, et al., Apoptosis in gastric mucosa with stress-induced gastric ulcers, J. Physiol. Pharmacol. 50 (1999) 211–225. [54] K.B. Hahm, I.S. Park, Y.S. Kim, J.H. Kim, S.W. Cho, S.I. Lee, et al., Role of rebamipide on induction of heat-shock proteins and protection against reactive oxygen metabolitemediated cell damage in cultured gastric mucosal cells, Free Radic. Biol. Med. 22 (1997) 711–716.

N

C

O

R

R

E

C

T

[3] A. Shiotani, Y. Yamaoka, H.M. El-Zimaity, M.A. Saeed, W.A. Qureshi, D.Y. Graham, NSAID gastric ulceration: predictive value of gastric pH, mucosal density of polymorphonuclear leukocytes, or levels of IL-8 or nitrite, Dig. Dis. Sci. 47 (2002) 38–43. [4] K. Sugano, T. Kontani, S. Katsuo, Y. Takei, N. Sakaki, K. Ashida, et al., Lansoprazole for secondary prevention of gastric or duodenal ulcers associated with long-term nonsteroidal anti-inflammatory drug (NSAID) therapy: results of a prospective, multicenter, double-blind, randomized, double-dummy, active-controlled trial, J. Gastroenterol. 47 (2012) 540–552. [5] M. Fornai, R. Colucci, L. Antonioli, O. Awwad, C. Ugolini, M. Tuccori, et al., Effects of esomeprazole on healing of nonsteroidal anti-inflammatory drug (NSAID)-induced gastric ulcers in the presence of a continued NSAID treatment: characterization of molecular mechanisms, Pharmacol. Res. 63 (2011) 59–67. [6] V. Lakshmi, N. Singh, S. Shrivastva, S.K. Mishra, P. Dharmani, V. Mishra, et al., Gedunin and photogedunin of Xylocarpus granatum show significant anti-secretory effects and protect the gastric mucosa of peptic ulcer in rats, Phytomedicine 17 (2010) 569–574. [7] D.D. Patra, A. Chattopadhyay, A.K. Singh, V.K.S. Tomar, A. Singh, H.O. Mishra, et al., Agrotechnology of kalmegh (Andrographis paniculatd), J. Med. Aromat. Plant Sci. 26 (2004) 534–537. [8] C. Xu, G.X. Chou, Z.T. Wang, A new diterpene from the leaves of Andrographis paniculata Nees, Fitoterapia 81 (2010) 610–613. [9] Y. Koteswara Rao, G. Vimalamma, C.V. Rao, Y.M. Tzeng, Flavonoids and andrographolides from Andrographis paniculata, Phytochemistry 65 (2004) 2317–2321. [10] P. Saranya, A. Geetha, S.M. Selvamathy, A biochemical study on the gastroprotective effect of andrographolide in rats induced with gastric ulcer, Indian J. Pharm. Sci. 73 (2011) 550–557. [11] S.G. Li, Q.S. Shao, Y.Y. Wang, T. Peng, Y.F. Zhao, Y.J. Yang, et al., Effect of andrographolide on proliferation and apoptosis of gastric cancer BGC-823 cells, Chin. J. Gastrointest. Surg. 16 (2013) 676–680. [12] X.W. Wang, F. Jiang, Z.W. Jing, F. Han, W.B. Rang, Clinical application and safety evaluation of lianbizhi injection, Chin. Med. Mod. Dist. Educ. China 10 (2012) 80–81. [13] Y. Ge, Y.X. Liu, L.L. Zhang SL, The chronic toxicity study on the adduct of andrographolide with sodium hydrosulfite in rats, Qilu Pharm. Aff. (2005) 562–564. [14] L.Y.D. Shi CY, Study on anti-inflammatory, antipyretic and analgesic effects of andrographolide sodium bisulfite, Chin. J. Ethnomed. Ethnopharm. (2009) 5–6. [15] H.M. Chen, H.J. Liao, Y.H. Liu, Y.F. Zheng, X.L. Wu, Z.Q. Su, et al., Protective effects of pogostone from Pogostemonis herba against ethanol-induced gastric ulcer in rats, Fitoterapia 100 (2015) 110–117. [16] W.F. Li, D.J. Hao, T. Fan, H.M. Huang, H. Yao, X.F. Niu, Protective effect of chelerythrine against ethanol-induced gastric ulcer in mice, Chem. Biol. Interact. 208 (2014) 18–27. [17] P.L. Beck, R. Xavier, N. Lu, N.N. Nanda, M. Dinauer, D.K. Podolsky, et al., Mechanisms of NSAID-induced gastrointestinal injury defined using mutant mice, Gastroenterology 119 (2000) 699–705. [18] R.F. Lin, X.X. Feng, C.W. Li, X.J. Zhang, X.T. Yu, J.Y. Zhou, et al., Prevention of UV radiation-induced cutaneous photoaging in mice by topical administration of patchouli oil, J. Ethnopharmacol. 154 (2014) 408–418. [19] Q.H. Zhang, C.F. Wu, L. Duan, J.Y. Yang, Protective effects of total saponins from stem and leaf of Panax ginseng against cyclophosphamide-induced genotoxicity and apoptosis in mouse bone marrow cells and peripheral lymphocyte cells, Food Chem. Toxicol. 46 (2008) 293–302. [20] B. Chowdhury, A.L. David, C. Thrasivoulou, D.L. Becker, D.L. Bader, T.T. Chowdhury, Tensile strain increased COX-2 expression and PGE2 release leading to weakening of the human amniotic membrane, Placenta 35 (2014) 1057–1064. [21] L. Fang, H.M. Chang, J.C. Cheng, P.C. Leung, Y.P. Sun, Nitric oxide and cGMP induce COX-2 expression and PGE2 production in human granulosa cells through CREB signaling pathway, J. Clin. Endocrinol. Metab. 100 (2015) E262–E269. [22] K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using realtime quantitative PCR and the 2(-delta delta C(T)) method, Methods 25 (2001) 402–408. [23] S.A. Bustin, V. Benes, J.A. Garson, J. Hellemans, J. Huggett, M. Kubista, et al., The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments, Clin. Chem. 55 (2009) 611–622. [24] H. Liu, F. Hou, H. Liang, G. Liu, Y. Li, Effects of diagnostic ultrasound on HSP70 expression in chorionic villi in rats during early pregnancy and the role of HSP70 in apoptosis in chorionic villi, Int. J. Mol. Med. 32 (2013) 1085–1092. [25] S.I. Abdelwahab, Protective mechanism of gallic acid and its novel derivative against ethanol-induced gastric ulcerogenesis: involvement of immunomodulation markers, Hsp70 and Bcl-2-associated X protein, Int. Immunopharmacol. 16 (2013) 296–305. [26] J. Dong, W. Liu, Y. Wang, Q. Xi, J. Chen, Hypothyroidism following developmental iodine deficiency reduces hippocampal neurogranin, CaMK II and calmodulin and elevates calcineurin in lactational rats, Int. J. Dev. Neurosci. 28 (2010) 589–596. [27] J. Li, Q. Ma, H. Liu, K. Guo, F. Li, W. Li, et al., Relationship between neural alteration and perineural invasion in pancreatic cancer patients with hyperglycemia, PLoS One 6 (2011) e17385.

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